February 7, 2019
Of all the mysteries surrounding the beginning of human life, one question has drawn particular interest from researchers at the Institute for Stem Cell and Regenerative Medicine (ISCRM). Simply put, for women who are unable to get pregnant, what is happening at the cellular level that is preventing life from taking hold?
Finding an answer may bring joy to millions of women and their partners around the world. According to the Centers for Disease Control, more than one in ten couples in the United States have trouble becoming pregnant[i].
Now, a paper in Nature Communications (Folliculin regulates mTORC1/2 and WNT pathways in early human pluripotency) details research that identifies a gene that may be complicit in many cases of human infertility. Dr. Julie Mathieu, an Assistant Professor at Comp Med and Director of ISCRM CRISPR-Core is the paper’s first author. Hannele Ruohola-Baker, Professor of Biochemistry and Associate Director of ISCRM, led the research team.
Ruohola-Baker is quick to emphasize that the study would not have been possible without several core capabilities that distinguish ISCRM as a world leader in stem cell-based research, including Dr. Mathieu’s skill with CRISPR[ii] technology, Yuliang Wang’s expertise with bioinformatics, and access to human stem cell lines developed by Dr. Carol Ware, Director of the Tom and Sue Ellison Stem Cell Core.
The study in Nature Communications, which builds on previous infertility research done at ISCRM, sought to expand current knowledge about the processes at work in the very early stages of embryonic development. Mathieu and Ruohola-Baker were especially focused on what might go wrong at one important step – implantation.
The meeting of the sperm and the egg initiates a chain of events that can generate a baby, but only if the embryo attaches to the mother’s uterus – the result of a successful implantation. Mathieu explains, “We know that in addition to the outer layer of the embryo, trophoblasts, the stem cells inside the blastocyst (a precursor to the embryo), play a key role in implantation. However, we know much less about the genes that control these stem cells.”
In a nutshell, which genes are important for fertility and how do these genes function?
To aid them in the investigation, Mathieu and Ruohola-Baker, in collaboration with Dr. Patrick Paddison at Fred Hutchinson Cancer Research Center, turned to the gene-editing system CRISPR to identify the genes that are involved in the stages of embryonic development that come just before and after implantation.
First, Mathieu designed a CRISPR screen to mutate genes suspected to play central roles in implantation and let them develop into a primed, or post-implantation, state. Next, she killed the cells that reached the post-implantation state, isolating the problem cells, and marking the genes that must be essential for implantation.
One gene, known as Folliculin(FLCN), caught the researcher’s attention. FLCN is a tumor suppressor associated with Birt Hogg Dube, a hereditary disorder that affects the kidneys and lungs. In her search for infertility culprits, Mathieu used CRISPR to knock out the FLCN gene in the pre-implantation stage, leading to an important revelation: When FLCN is mutated, the embryo stem cells do not advance to the post-implantation stage.
Mathieu explains the importance of this finding. “In cells that have this mutation in FLCN, we saw a transcription factor, called TFE3, in the nucleus later in the development process than it should be. We believe TFE3’s presence in the nucleus is preventing the cells from exiting the pre-implantation state by keeping the Wnt pathway active. We discovered that by inhibiting this pathway, we could rescue the FLCN mutant phenotype, allowing the embryonic stem cells to move to a post-implantation state.”
There was another clue pointing to FLCN. In their research, Mathieu and Ruohola-Baker collaborated with the Randall T. Moon Laboratory, named for the Founding Director of ISCRM, to identify proteins that bind to FLCN. They found FLCN in different protein complexes before and after implantation – revealing the molecular mechanism for this important protein. Critical proteins associated with FLCN, known as mTor regulators, may also point scientists to new treatments for cancer, kidney disease, and lung collapse.
Understanding the nature of a gene that plays such a critical role in the implantation process brings the ISCRM team one step closer to addressing the biggest cause of infertility, giving hope to would-be parents struggling to get pregnant.
The study was funded by the National Institutes of Health (R01GM097372, R0GM97372-0351, and R01GM083867, 1P01GM081619) and the National Institutes of Health Progenitor Cell Biology Consortium (U01Hl099997, U01HL099993), and a UW Medicine Stem Cell and Reproductive Medicine Pilot Award.
The stem cell lines for this study were developed by Carol Ware, professor of comparative medicine at the UW School of Medicine, and director of the Tom and Sue Ellison Stem Cell Core Facility. Yuliang Wang, research assistant professor of computer science and engineering at the UW Paul G. Allen School, led the project’s computational biology aspects. The Feb. 7 Nature Communications paper, “Folliculin regulates mTORC1/2 and WNT pathways in early human pluripotency,” provides a full list of collaborators for this project.
[i] About one-third of infertility cases are caused by fertility problems in women, and another one-third of fertility problems are due to fertility problems in men. The other cases are caused by a mixture of male and female problems or by problems that cannot be determined.
[ii] CRISPR is a gene editing system that allows researchers to target specific stretches of genetic code and to edit DNA at precise locations